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Abstract

Background and objectives

Breast cancer is one of the most common causes of cancer-related deaths in women worldwide.
Studies on glucosylceramide synthase (GCS) activity suggest that this enzyme has a
role in the development of multidrug resistance in many cancer cells. However, few
studies have shown the expression of GCS in invasive ductal breast cancer and breast
intraductal proliferative lesions.

Methods

In total, 196 samples from patients with invasive ductal breast cancer and 61 samples
of breast intraductal proliferative lesions were collected. Immunohistochemical analyses
were conducted to determine the expression of GCS and other related proteins.

Results

Expression of GCS was high in estrogen receptor (ER)-positive and HER-2 negative samples.
In contrast, the expression of GCS in invasive ductal cancer was significantly lower
than that in intraductal proliferative lesions.

Conclusion

Our data demonstrates a correlation between the expression of the GCS protein and
ER-positive/HER-2 negative breast cancer. Furthermore, in contrast to previous reports,
the expression of GCS protein was shown to be much higher in ductal carcinoma in-situ
than that in invasive ductal cancer.

Keywords:

Background

Breast cancer is one of the most common causes of cancer deaths in women worldwide
[1]. The prognosis of breast cancer has been improved significantly by comprehensive
therapy including surgical methods, chemotherapy, endocrine therapies and molecular
targeted therapy. However, multidrug resistance (MDR) has been a major barrier to
improved survival rates among breast cancer patients. MDR refers to the resistance
of tumors not only to individual cytotoxic drugs used in chemotherapy, but also to
cross-resistance to a range of drugs with different structures and cellular targets
[2].

P-glycoprotein (P-gp, P170), encoded by the MDR1 gene (ABCB1) in humans is the major cause of multidrug resistance in breast cancer. P-gp is a
member of the adenosine triphosphate-binding cassette (ABC) superfamily of membrane
transporters, which bind and hydrolyze ATP. The energy produced in this reaction is
used to drive the active transport of various molecules across the plasma membrane
or the intracellular membranes of organelles, such as the endoplasmic reticulum, peroxisomes,
and mitochondria. A wide range of anticancer agents are actively extruded by P-gp,
leading to chemoresistance [3].

Many studies have indicated that MDR1 is regulated by glucosylceramide synthase (GCS),
which is a pivotal enzyme in the regulation of cellular ceramide [4]. Studies on GCS activity suggest that the enzyme plays a role in the development
of MDR in many cancer cells [5,6]. A number of methods that suppress the expression of GCS, such as specific inhibitors,
antisense oligonucleotides and siRNA, have been shown to render MDR cells chemosensitive
[7,8]. Gouaze et al. suggested that GCS blockade resensitizes MDR breast cancer cells to
anticancer drugs by downregulation of P-glycoprotein [9]. Liu et al. further demonstrated that GCS upregulates MDR1 expression to regulate
cancer drug resistance through cSrc and beta-catenin signaling [10].

Few studies have shown the expression of GCS in breast cancer tissue samples. In 2009,
Ruckhäberle et al. analyzed microarray data that showed GCS mRNA expression levels
in 1,681 breast tumors [11], but few data have demonstrated the expression of the GCS protein in breast cancer.
In 2011, Liu et al. detected GCS expression levels in normal tissues and certain cancer
tissues; however, this investigation was conducted in only a small number of samples
[12]. Zhang et al. showed the relationship between chemotherapy and GCS expression in
invasive breast cancer tissue. However, there are currently no reports describing
the expression of GCS in clinical samples of intraductal proliferative breast lesions.
This study aimed to rectify this omission.

Methods

Clinical samples

Tissue samples from 257 patients who underwent complete dissection of the breast and
axillary lymph nodes (breast cancer patients) or local lumpectomy (patients with intraductal
proliferative lesions or patients with accessory breast) were collected at the Qilu
Hospital and Provincial Hospital, Shandong University, China, between January 2006
and June 2010. No patients had preoperative chemotherapy and informed consent for
pathological evaluation was obtained from all patients prior to surgery.

The use of these tissues was approved by the Research Ethics Committee of Shandong
Medical University and we obtained written informed consent from all participants
involved in our study.

Cell culture

The multidrug-resistant breast cancer cell line, MCF-7/ADM, was selected from the
drug sensitive breast cancer cell line MCF-7 using Doxorubicin in stages. Cells were
maintained in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS)
in a humidified atmosphere containing 5% CO2 at 37°C. Cells were then seeded on glass slides for 24 h. Overexpression of GCS protein
by MCF-7/ADM cells was confirmed for use of these cells as a positive control in this
study [13].

Morphologic parameters

Two pathologists, an experienced senior pathologist and a less experienced junior
pathologist reevaluated all of the tumor slides stained with hematoxylin and eosin
(HE) for the following morphological features and the histological tumor type according
to WHO 2003 classification. The morphological features were categorized into 3 groups
[14]:

1. Grading factors: Histological grade was assessed using the modified Bloom-Richardson
method, in which tubule formation/grade of the tumor, nuclear pleomorphism/atypia
(nuclear grade), mitotic count were scored. Mitotic count was performed on Olympus
BX51 light microscope, with a graticule at × 40 magnification and in 10 high-power
fields (HPFs). Mitotic number was scored as 1 when it was between 0–7, 2 when between
8–14 and 3 when 15 or more.

2. Architectural features of the tumor:

i. Tumor growth pattern was assessed as infiltrative if there was irregular infiltration
into the surrounding parenchyma or fat or pushing if the tumor was well circumscribed.

ii. Necrosis with its type was noted as present or absent. Large confluent areas of
tumor necrosis with an irregular outline called as geographicnecrosis and the necrosis
in the middle of the tumor islands was called as central necrosis.

iii. Stromal lymphocytic response was scored as none, mild (less than 25% of the tumor),
moderate (25 to 50% of the tumor) and marked (>50% of the tumor).

i. Presence of nucleoli were scored as absent or prominent if they were easily visible
at low power

ii. Amount of the tumor cell cytoplasm was assessed as scant, moderate or copious
according to nuclear-cytoplasm ratio.

iii. Presence of vesicular chromatin pattern was noted.

Immunocytochemical and immunohistochemical analyses

Immunohistochemical staining was carried out using the DAKO Envision detection kit
(Dako, Carpinteria, CA, USA). In brief, paraffin-embedded tissue blocks were sectioned
(4 μm-thick), dried, deparaffinized, and rehydrated. Antigen retrieval was performed
in a microwave oven for 15 min in 10 mM citrate buffer (pH 6.0). After cells were
embedded in 4% neutral formaldehyde for 2 h, PBS with 0.5% Tween-20 was added for
30 min at room temperature. For all samples, endogenous peroxidase activity was blocked
with a 3% H2O2-methanol solution. The slides were blocked with 10% normal goat serum for 10 min
and incubated with an appropriately diluted primary antibody overnight at 4°C. The
slides were then probed with an HRP-labeled polymer conjugated to an appropriate secondary
antibody for 30 min. The antibodies against estrogen receptor (ER), progesterone receptor
(PR), HER-2, Ki67, cytokeratin 5/6 (CK5/6) and epidermal growth factor receptor (EGFR)
were purchased from Dako (Carpinteria, CA, USA) and the GCS antibody was a gift from
Dr. D. Marks (Mayo Clinic Center).

Interpretation

Staining results were interpreted by a breast pathologist who was blinded to patient
outcomes. Tumors with 1% or more positively stained nuclei were considered positive
for ER and PR expression [15]. Ki67 staining was determined to be positive when more than 14% of the nuclei were
stained. Membranous staining for EGFR and cytoplasmic staining for CK5/6 and HER-2
were scored by counting the number of positively stained cells on the membrane and
expressed as a percentage of total tumor cells according to the American Society of
Clinical Oncology (ASCO) and the College of American Pathologists (CAP) guidelines
using the following categories: 0, no immunostaining; 1+, weak, incomplete membranous
staining in any proportion of tumor cells; 2+, complete membranous staining, either
non-uniform or weak in at least 10% of tumor cells; and 3+, uniform, intense membranous
staining in >30% of tumor cells. HER-2 results were considered positive in cases with
3 + membranous staining of IHC or gene amplification by fluorescence in-situ hybridization
(FISH) irrespective of IHC results using the diagnostic criteria described [16].

A dual semi-quantitative scale combining staining intensity and percentage of positive
cells was used to evaluate GCS protein staining. The staining intensity was scored
as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). The percentage of positive
cells was scored as follows: 0, no staining or staining in <5% of tumor cells; 1,
staining in 5% to 25% of cells; 2, staining in 26% to 50% of cells; 3, staining in
51% to 75% of cells; and 4, staining in >75% of cells. For GCS, cytoplasmic staining
was considered positive, with an IHC score ≥2 defined as high expression and <2, as
low expression [13].

Fluorescence in-situ hybridization

In cases of HER-2 IHC staining of 2+, fluorescence in-situ hybridization (FISH) analysis
was performed manually using the PathVysion HER-2 DNA Probe Kit (Abott, Abott Park,
IL, USA) according to the manufacturer’s instructions. In brief, consecutive sections
from formalin-fixed, paraffin-embedded tissue blocks were mounted on Probe On Plus
microscope slides (Fisher Scientific, Pittsburgh, PA, USA), deparaffinized in xylene,
and subsequently rehydrated in ethanol. Sections were then boiled for 10 min in pretreatment
solution, incubated with pepsin solution for 10 min, dehydrated in ethanol for 6 min,
and finally air-dried. For hybridization, the buffered probe (HER-2/neu and centromere
17) was added to the slide and protected by a coverslip that was sealed with rubber
cement. For denaturation, slides were heated to 82°C and incubated overnight at 45°C
in a dark, humidified chamber. The rubber cement and coverslip were then removed,
and the slides were transferred to stringent wash buffer for 10 min at 65°C. Sections
were then dehydrated in ethanol for 6 min and air-dried before being counterstained
with 40, 6-diamidino-2- phenylindole (DAPI). Evaluation of signals was carried out
using an epifluorescence microscope (Leica, Germany). Counting was carried out according
to the manufacturer’s instructions (HER-2/neu gene, orange; centromere 17, green).
As recommended by the ASCO/CAP guidelines, an absolute HER-2 gene copy number greater
than 6 or HER-2 gene/chromosome 17 copy number ratio higher than 2.2 was considered
HER-2 positive. Lymphocytes, fibroblasts, and normal ductal epithelial cells were
used as internal controls.

Statistical analysis

Chi-square or Fisher’s exact tests was used to analyze the relationship between the
expression of GCS and each histopathological variable. Survival curves were plotted
using the Kaplan-Meier method and were compared using the log-rank test. P-values less than 0.05 were considered statistically significant. All calculations
were performed using the SPSS16.0 for windows statistical software package (SPSS,
Chicago, IL, USA).

Results

Expression of GCS protein in breast tissue samples

The expression of GCS protein was detected in all samples by immunohistochemical staining.
Figure 1 show positively and negative staining in invasive ductal breast cancer of breast
samples (Figure 1 and Table 3).

Figure 1.Immunohistochemical analyses of GCS protein in different sample types. GCS protein expression detected in all samples by immunohistochemical and cytoplasmic
staining was considered positive. Images are representative of two cases that were
predominantly positive in DCIS (A) or IDC (B), respectively. DCIS-ductal carcinoma in situ; IDC-invasive ductal carcinoma.

Table 3.The correlation between GCS and the histopathological variables in 143 cases of invasive
breast cancer

Correlation between GCS expression and clinicopathological parameters

Overall, 72.7% of all the invasive carcinoma samples were positive for GCS (104/143),
while only 93.4% of the intraductal proliferative lesions were positive (57/61). The
expression level of the GCS protein in the intraductal proliferative lesions was significantly
higher than that in the invasive ductal carcinoma (P < 0.05).

In the invasive cancers, there was a significant correlation between the GCS upregulation
and ER positivity (P = 0.017) or HER-2 negativity (P = 0.007). We also found that positive rates of GCS expression were higher in grade
I (93.75%, 15/16) than those in the grade II–III (70.08%, 89/127) (P = 0.045) (Table 3). A higher positive rate of GCS expression was observed in younger patients (aged
<35 years) compared with that in older patients (aged ≥35 years).

There was no statistical significance in the relationship between GCS expression and
other clinicopathological parameters, including age, tumor size, nodal stage, Ki67
(Table 3).

Correlation between the GCS expression and the survival

There was no statistical significance in the relationship between GCS expression and
overall survival (Figure 2).

The positive rate of GCS was highest in luminar A tumors and was lowest in basal-like
tumors. However, there was no statistically significant difference in the GCS expression
levels between the four breast tumor subtypes (Figure 3).

Discussion

Breast cancer is one of the most frequent and deadly cancers in women, with an estimated
1,300,000 new cases and 465,000 deaths annually [17]. Multidrug resistance is one of the main impediments to the successful treatment
of breast cancer. The mechanisms underlying MDR are complex and overexpression of
P-gp is considered to be an important factor.

Recent research has indicated that the expression of P-gp is related to the activity
of GCS, an enzyme that glycosylates ceramide and inhibits its proapoptotic activity
in cells. Zhang et al. revealed that the expression of the GCS gene in the drug-resistant
human breast cancer cell line MCF-7/ADM is higher than that in drug sensitive cells,
and that the sensitivity of MCF-7/ADM cells to adriamycin is enhanced by GCS inhibition
[18]. Furthermore, GCS expression has been found to confer MDR in many other cancers [19,20]. MDR1 and GCS have been shown to be overexpressed coincidently in several drug-resistant
cell lines, a phenomenon that indicates a relationship between these two proteins.
In 2010, Liu et al. demonstrated for the first time that GCS upregulates MDR1 expression
resulting in the modulation of drug resistance in the ovarian drug-resistant cell
line NCI/ADR-RES through the cSrc and beta-catenin signaling pathway [10].

In 2009, microarray analysis of 1,681 breast tumors conducted by Ruckhäberle et al.
revealed that GCS mRNA expression was associated with positive ER status, lower histological
grading, low Ki67 levels and ErbB2 negativity (P < 0.001 for all) [8]. In 2011, Liu et al. detected GCS expression levels in normal tissues and certain
cancer tissues. Their results showed that GCS overexpression is highly associated
with ER-positive and HER-2-positive breast cancers that have metastasized [12]; however, this was a small study. Our results demonstrated that GCS protein expression
was higher in ER-positive samples (P < 0.05) (Table 3), which was in accordance with both of these previous studies.

Human epidermal growth factor receptor 2 (HER2) protein, encoded by the oncogene HER2,
is amplified in 20–30% of breast cancer cases and is the target of HER2-directed anti-cancer
therapies [21]. Our research shows that there was a significant correlation between GCS expression
and low HER-2 status in the invasive ductal cancer samples (Table 3), which was in accordance with the study of Ruckhäberle et al., although our observation
that GCS protein levels did not correlate with Ki67.

Our study demonstrated a higher positive rate of GCS expression in breast cancer samples
from younger patients (aged <35 years) expressed lower levels of GCS protein than
older patients (aged ≥35 years) (60% vs. 74.8%, P = 0.035). Otherwise, we found that the expression of GCS was higher in the cancer
T1-2 than that in the cancer T3-4.

Breast cancer is accounting for 23% (1.38 million) of the total new cancer cases and
14% (458,400) of the total cancer deaths in 2008 worldwide. Metastasis and recurrence
severely affect the quality and length of lives of breast cancer patients [22]. Although the study of Liu demonstrated that GCS overexpression is highly associated
with ER-positive and HER-2-positive breast cancers that have metastasized [12], our study demonstrated that GCS expression has no correlation with lymph metastasis.

Our data also showed that, in contrast to previous reports, GCS protein expression
was much higher in DCIS than that in the invasive ductal cancer.